The present invention relates to the chemical derivatization of amino acids. More specifically, a method is disclosed for the preparation of fluorescent derivatives of amino acids suitable for analysis and detection using liquid chromatography with reversed phase separation columns. In general the method can be applied to the analysis of amino acids in a variety of matrices such as physiological fluids, pharmaceuticals, foods and beverages, etc.
Today, in many technical areas, the analysis of amino acids is an important requirement. Particularly in the rapidly growing area of biotechnology, this analysis is a critical procedure in the characterization of proteins and peptides. In addition, amino acid analysis is required in the medical field, where characterization of amino acids in biological materials can be useful in diagnostic procedures; in the pharmaceutical field for development and quality control of products; and in the food and beverage area, again for product characterization.
For successful amino acid analysis, typically between 18 and 35 amino acids (depending on the matrix) must be separately quantified in amounts typically between 100 femtomoles (10.sup.-13 moles) and 1 nanomole (10.sup.-9 moles) each, preferably with less than 5% sample to sample relative standard deviation, often in the presence of other matrix components, and preferably in less than 1 hour per sample. Modern developments favor high sensitivity and fast analyses.
Most analytical procedures for amino acids are based on liquid chromatography, either in its modern high performance form (HPLC), or as classical medium pressure liquid chromatography. In either case, the major problem in the analysis is the selective and sensitive detection of these compounds. With few exceptions, the amino acids do not show strong optical absorption above 220 nm. This precludes the use of ultraviolet/visible spectrophotometric detection at the required sensitivity. Similarly, detection based on refractive index lacks the required sensitivity. For these reasons, chemical derivatization procedures are generally used. The chemical derivatizations tag the compounds with a chemical group so that the resulting product has strong response for either UV/VIS or Fluorescence detection. The derivatization procedure can be performed prior to the chromatographic separation ("precolumn") or after the separation ("postcolumn").
In most cases, the amino group of the amino acid is used as the active site for the chemical derivatization, but because of the diverse chemical nature and reactivity of the various amino acids, the analysis still is not of uniform exactness. Around a common backbone, the amino acids contain such differing functional groups as aliphatic, aromatic, primary amines, secondary amines, carboxylic acids, amides and thiols. This makes it difficult to find a reagent which will react with all amino acids and yield comparable sensitivity.
Current chromatographic methods for amino acid analysis can be classified on the basis of the derivatization mode (precolumn or postcolumn) or the detection mode (UV/VIS or Fluorescence) (Amino Acid Analysis, J. M. Rattenbury ed. (Wiley Interscience, New York, 1981)).
In terms of the derivatization mode, precolumn derivatization is becoming increasingly more popular because it allows the use of high efficiency, small particle, reversed phase chromatographic columns. Postcolumn derivatization can be used only after separation of the amino acids using ion exchange chromatography, which tends to exhibit poorer chromatographic efficiency and longer analysis time.
The most popular UV/VIS derivatization reactions for amino acids employ either ninhydrin (S. Moore, D. H. Spackmann and W. H. Stein, Anal. Chem. 30 (1958) 1185-1205) or PITC (phenylisotHiocyanate) (R. L. Hendrickson and S. C. Meredith, Anal. Bioch., 136 (1984) 65-74). UV/VIS derivatization, however, has relatively low detection sensitivity compared to fluorescence detection. Ninhydrin can be used only postcolumn, but allows the detection of all amino acids. PITC is run precolumn and allows detection of all amino acids, but requires a derivatization procedure which is not amenable to automation.
The most popular fluorescence derivation reactions utilize either OPA (ortho-phthalaldehyde) (P. Lindroth and K. Mopper, Anal. Chem. 51 (1979) 1667 and M. Roth, Anal. Chem. 43/1971) 880); FMOC (Fluorenylmethylchloroformate) (S. Einarsson, B. Josefsson and S. Lagerkvist, J. Chromatogr. 292 (1983) 609-618); or Dansyl Chloride (Y. Tapuhi, D. E. Schmidt, W. Lindner and B. L. Karger, Anal. Bioch. 15 (1981) 123). OPA is based on a simple, fast, easily automated procedure with high sensitivity, but reacts only with primary amines. Current methods usually react OPA in the presence of mercaptoethanol which results in reagent and products with limited stability. Both FMOC and Dansyl Chloride are used precolumn, deliver high sensitivity, and react with primary and secondary amines, but require long reaction times and ancillary procedures, such as extractions, to remove excess reagent.
The simultaneous detections of both cystine and cysteine have presented particular problems in the past for the aforesaid methods. Cystine is a molecule composed of two cysteine molecules joined by a disulfide bridge. Generally, a derivative of either cystine or cysteine can be detected by the prior art, but the substantial interconversion of one to the other that can often take place, depending on the sample history, lowers the accuracy of the analysis for either.